Live attenuated vaccine 17D has been used since the 1950s for vaccination against yellow fever (YF) with a remarkable record of safety and efficacy. More than 500 million people have been vaccinated, and the World Health Organization (WHO) strongly recommends to continue vaccinations in at-risk countries. The weaknesses of the vaccine include outdated manufacturing and the need of a "cold chain", which accounts for up to 80% of cost in endemic areas. In rare cases, 17D vaccine causes adverse effects including allergies, neurologic disorders, and viscerotropic disease. The vaccine represents a population of genetically distinct viruses, some of which may be responsible for adverse reactions. The main goal of this revised Phase I SBIR is the production and evaluation of a conceptually novel YF vaccine. We propose a novel technology of infectious DNA (i-DNA) as YF vaccine. A unique feature of this technology is that the full-length copy of 17D genome is placed in the i-DNA plasmid in the context of optimized eukaryotic promoter and regulatory sequences. Thus, live attenuated 17D virus can be launched in vivo directly from the i-DNA plasmid. Since the 17D i-DNA represents a molecular clone, it will generate a uniform population of 17D virus thus potentially improving safety. We will also prepare two i-DNA variants by de-optimization of translational codons within C-prM-E genes with the view to improve vaccine safety and genetic stability. Experimental YF i-DNA vaccines will be evaluated in vitro and in vivo along with the current 17D vaccine. Immunogenicity and safety profiles including neurotropic and viscerotropic adverse effects will be evaluated in the recently developed models of immunosupressed hamster and A129 knockout mouse. Thus, characteristics of candidate i-DNA vaccines will be evaluated in the two preclinical models with immunocompromised background, which mimicks frequent situation in the endemic areas and will provide accurate determination of safety and immunogenicity profiles of the vaccines. In summary, i-DNA vaccination combines the simplicity of DNA vaccines with the exceptional efficacy of live attenuated vaccines. The i-DNA can potentially improve safety, does not require cold chain and is easy to manufacture and scale-up in emergency scenarios. Further, bacterially generated i-DNA will contain CpG motifs, which are expected to activate innate immune responses and improve immunogenicity. If successful, this technology may represent a revolutionary improvement of YF vaccine and vaccination practice against yellow fever. PUBLIC HEALTH RELEVANCE: Yellow fever (YF) is a re-emerging pathogen and a public health problem worldwide. The focus of this Phase I SBIR study is the production and evaluation of a conceptually novel vaccine for YF. We hypothesize that safety and immunogenicity of live attenuated 17D vaccine can be improved by using the infectious DNA (i-DNA) technology. This will result in a unique YF vaccine, which will combine the simplicity of DNA vaccines with the exceptional efficacy of live attenuated vaccine.